Characterization and enzymatic degradation of a cross-linked bacterial polyester

The bacterial polyester, poly(-hydroxybutyrate-co--hydroxyvalerate) (PHB/V), was cross-linked with 1, 5, 7, 10, 20, and 30 wt% benzoyl peroxide by thermal decomposition reactions. Solvent extractions were carried out to determine the cross-linked fractions of the films. The sol/gel data were used to estimate cross-link densities. Films of PHB/V cross-linked with 10% benzoyl peroxide were placed in contact with purified depolymerase A secreted byP. lemoignei. These samples exhibited weight loss rates which were half that of un-cross-linked PHB/V, but the network was degraded completely by the enzyme. The results of this study suggest that anendo-type enzymatic degradation may occur, in addition to theexo-type activity, which is normally presumed to occur with theP. lemoignei depolymerase system.     

Biodegradation of starch-poly(β-hydroxybutyrate-co-valerate) composites in municipal activated sludge

Injection-molded composites were prepared by blending PHBV⁵ with native cornstarch (30% and 50%) and with cornstarch precoated with PEO as a binding agent. These composites were evaluated for their biodegradability in municipal activated sludge by measuring changes in their physical and chemical properties over a period of 35 days. All composites lost weight, ranging from 45 to 78% within 35 days. Interestingly, the extent and rate of weight loss were quite similar in PHBV composites with no starch, with 30% starch, and with 50% starch. Weight loss was slowest in PHBV blends prepared with PEO-coated starch. For all samples, the weight loss was accompanied by a rapid deterioration in tensile strength and percentage elongation. The deterioration of these mechanical properties exhibited a relative rate of PHBV>starch-PHBV>PEO-coated starch-PHBV. Changes in starch/PHBV composition after biodegradation were quantified by FTIR spectroscopy. Increasing the starch content resulted in more extensive starch degradation, while the PHBV content in the blends became less susceptible to hydrolytic enzymes.The mention of firms names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over firms or similar products not mentioned. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, marital status, or handicap.     

Assessing the biodegradation potential of polymers in screening- and long-term test systems

This paper presents a test scheme for assessing the biodegradation potential of polymers, starting with aquatic screening systems (aerobic and anaerobic) and continuing to long-term systems. At the end of the scheme the material has to prove its behavior under the relevant disposal conditions. Aerobic screening was performed mainly under aquatic conditions, but also in soil, using BOD-respirometry. Carbon balances were performed to obtain a better evaluation of the biodegradation potential. Under anaerobic conditions, biodegradation in an aquatic medium was followed by measuring CH₄ and CO₂ production. Polymers not fully degraded in the screening systems were tested in aquarium systems for at least 1 year. Biodegradation was followed by monitoring the DOC released in the water, mass loss, and microbial growth on the samples and in the water as well as via FTIR spectroscopy and SEM pictures. Results are presented for the polymers PHB, PHBV, PCL, Mater-Bi AI05H and ZF03U, and Bioceta. By combining the data from the screening with the aquarium system, a good picture of the degradation behavior of the polymers is obtained.Paper presented at the Bio/Environmentally Degradable Polymer Society—Third National Meeting, June 6–8, 1994, Boston, Massachusetts.     

The Effect of UV-B Irradiation on the Biodegradability of Poly-β-Hydroxybutyrate (PHB) and Poly-ɛ-Caprolactone (PCL)

The biodegradability of poly--hydroxybutyrate and poly--caprolactone in soil compostage before and after irradiation of the polymers for 192, 425, and 600 h in a Weather-Ometer was examined. The biodegradability tests were done in soil compostage at pH 7.0, 9.0, and 11.0 to assess the influence of this parameter on degradation. The rate of degradation was directly proportional to the soil alkalinity. Poly--hydroxybutyrate showed the greatest weight loss and aging in a Weather-Ometer did not significantly increase the biodegradation, except when the polymer was aged for 425 h and buried in soil compostage of pH 11.0.     

Distribution of Poly(β-propiolactone) Aerobic Degrading Microorganisms in Different Environments

The distribution of degading microorganisms of high molecular weight poly(-propiolactone) (PPL), whose individual structural units are similar to those of poly(-hydroxybutyrate) (PHB) and poly(€-caprolactone) (PCL), was examined. Despite the fact that PPL is a chemosynthetic polymer, many kinds of PPL-degrading microorganisms were found to be distributed as resident populations widely in natural environments. A total of 77 strains of PPL-degrading microorganisms was isolated. From standard physiological and biochemical tests, at least 41 strains were referred to as Bacillus species. Microbial degradation of fibrous PPL proceeded rapidly in some enrichment cultures but was not as complete as that of PHB. Most of the isolated PPL-degrading microorganisms were determined to be PCL degraders and/or PHB degraders. Therefore, it can be assumed that mostly PPL is recognized by the microorganisms as PHB or another natural substrate of the same type as which PCL is regarded. Microbial degradation of PPL was confirmed by some Bacillus strains from type culture collections. The similarity of microbial degradation between PPL and PCL was found to be very close.     

Distribution of poly(β-hydroxybutyrate) and poly(ε-caprolactone)aerobic degrading microorganisms in different environments

To assess the capacity of the natural environment for degrading plastics, the populations of poly(-hydroxybutyrate)(PHB)-and poly(-caprolactone)(PCL)-degrading aerobic microorganisms and their ratios to the total number of microorganisms in soil samples were estimated by the plate count method with agar medium containing emulsified PHB or PCL. The numbers of the degrading microorganisms were determined by counting colonies that formed clear zones on the plate. It was found that PHB- and PCL-degrading (depolymerizing) microorganisms are distributed over many kinds of material, including landfill leachate, compost, sewage sludge, forest soil, farm soil, paddy soil, weed field soil, roadside sand, and pond sediment. Of total colony counts, the percentages of PHB and PCL degrading microorganisms were 0.2–11.4 and 0.8–11.0%, respectively. The results suggest that many kinds of degrading microorganisms are present in each environment and that specific consortia differing in biodegradation capacity are constructed.     

Degradation of poly(β-hydroxyalkanoates) and polyolefin blends in a municipal wastewater treatment facility

Six types of plastics and plastic blends, the latter composed at least partially of biodegradable material, were exposed to aerobically treated wastewater (activated sludge) to ascertain their biodegradability. In one study, duplicate samples of 6% starch in polypropylene, 12% starch in linear low-density polyethylene, 30% polycaprolactone in linear low-density polyethylene, and poly(-hydroxybutyrate-co-hydroxyvalerate) (PHB/V), a microbially produced polyester, were exposed to activated sludge for 5 months, and changes in mass, molecular weight average, and tensile properties were measured. None of the blended material showed any sign of degradation. PHB/V, however, showed a considerable loss of mass and a significant loss of tensile strength. In a second study, PHB/V degraded rapidly, but another type of microbial polymer which forms a thermoplastic elastomer, poly(-hydroxyoctanoate), did not degrade. These results illustrate the potential for disposal and degradation of PHB/V in municipal wastewater.     

Synthesis of Poly(β-hydroxybutyrate) in Simulated Microgravity: An Investigation of Aeration Profiles in Shake Flask and Bioreactor

The microbial strain Azotobacter vinelandii UWD was grown under conditions of simulated microgravity in the National Aeronautics and Space Administration (NASA) Bioreactor. Bacterial growth in simulated microgravity differed significantly from that observed in conventional shake flask experiments: Cells tended to grow in a cluster-like pattern and polymer production started immediately after exposing them to conditions of simulated microgravity, and no lag time was observed. It was imperative to differentiate between the effects derived from microgravity and those imposed by the altered oxygen supply in the bioreactor. Aeration conditions were studied in both reactor types and a gas supply profile was developed for the bioreactor. This supply profile allowed for similar amounts of dissolved oxygen in the bioreactor and the shake flask in the initial stage of the fermentation and, therefore, for an evaluation of the effects of microgravity on biopolyester-producing bacteria. Since the optical density that is conventionally used as a measure for the cell growth could not be used due to the cluster-like growth pattern of the cells, it was determined that bacterial growth behavior in the bioreactor can be monitored through glucose or oxygen consumption.     

Enzymatic degradation of poly([R,S] β-hydroxybutyrate)

The synthetic analogue of a bacterially produced polyester, poly(-hydroxybutyrate) (PHB) was synthesized from racemic -butyrolactone using anin situ trimethyl aluminum-water catalyst. The polymer was fractionated into samples differing in molecular weight and isotactic diad content. The latter was closely related to degree of crystallinity. The biodegradation of these fractions were examined by monitoring mass loss over time in the presence of anAlcaligenes faecalis T1 extracellular bacterial poly(-hydroxybutyrate) depolymerase. The fraction with high isotactic diad tacticity content showed little or no degradation over a 50 hour incubation period, whereas the fraction of intermediate isotactic diad content degraded in a continuous steady fashion at a rate that was less than that for bacterial PHB. The low isotactic diad fraction underwent a rapid initial degradation, followed by no further mass loss. The presence of stereoblocks in the polymer structure of the various fractions was an influence on the degree of susceptibility towards degradation and is related to sample crystallinity.     

A method for the isolation of microorganisms producing extracellular long-side-chain poly (β-hydroxyalkanoate) depolymerase

A simple method was developed for the preparation of an autoclavable, long-side-chain poly (-hydroxyalkanoate) (LSC-PHA) colloidal suspension, which was used as a substrate for enzymatic degradation and to prepare agar overlay plates for the isolation of microorganisms producing extracellular LSC-PHA depolymerase. Six cultures producing extracellular LSC-PHA depolymerase were isolated from a composted hydrocarbon-contaminated soil. All were pseudomonads or related bacteria. All (with the possible exception ofXanthomonas maltophilia) could produce LSC PHA. Except forX. maltophilia none could hydrolyze poly (-hydroxybutyrate). Screening of sevenPseudomonas strains known to accumulate LSC PHA showed that all were negative for extracellular LSC-PHA depolymerase production. It was concluded that extracellular LSC-PHA depolymerase producers are found mostly in the genusPseudomonas but that they are relatively uncommon.     

Preparation conditions and swelling equilibria of biodegradable hydrogels prepared from microbial poly(γ-glutamic acid) and poly(ε-lysine)

Biodegradable hydrogels prepared by -irradiation from microbial poly(amino acid)s are reviewed. pH-sensitive hydrogels were prepared by means of -irradiation of poly(-glutamic acid) (PGA) produced byBacillus subtilis IFO3335 and poly(-lysine) (PL) produced byStreptomyces albulus in aqueous solutions. The preparation conditions, swelling equilibria, hydrolytic degradation, and enzymatic degradation of these hydrogels were studied. A hydrogel with a wide variety of swelling behaviors has been produced by -irradiation from a mixture solution of PGA and PL.Paper presented at the 4th International Workshop on Biodegradable Plastics and Polymers, October 11–14, 1995, Durham, New Hampshire, USA.     

Biodegradability and compostability of polymers—test methods and criteria for evaluation

The treatment of solid waste in controlled composting facilities is an important possibility for reducing garbage. Natural and synthetic polymeric materials can be used for many purposes, for example, as packaging materials, where compostability is required. A prerequisite for official regulations and the decision as to which materials may be composted is investigations on their biodegradability and the quality of the compost produced. Several standardization groups at the ISO, CEN, and DIN are developing definitions, test methods, and classification systems for differentiating compostable from noncompostable materials. The concept which will be standardized and used in Germany is described in detail. It includes characterization of the test material, determination of the biodegradability using laboratory tests such as simple aquatic batch tests and a controlled aerobic composting test, investigation of the disintegration of the test material in industrial or bench-scale composting facilities, and finally, chemical and ecotoxicological analysis of the compost produced.     

The degradation of gel-spun poly(β-hydroxybutyrate) “wool”

The apparent biodegradability and biocompatibility of the microbially produced polyester, poly(-hydroxybutyrate) (PHB), has been the focus of much research by a number of authors with regard to its potential for use in packaging and medical implantation devices. PHB has recently been produced by gel-spinning into a novel form, with one possible application being as a wound scaffolding device, designed to support and protect a wound against further damage while promoting healing by encouraging cellular growth on and within the device from the wound surface. This new nonwoven form combines a large volume with a low mass, has an appearance similar to that of cotton wool, and has been called wool because of this similarity. The hydrolytic degradation of this wool was investigated in an accelerated model of pH 10.6 and temperature 70°C. It was determined that the PHB wool gradually collapsed during degradation. The surface area-to-volume ratio was concluded to be a primary influencing factor. Degradation was characterized by a reduction in the glass transition temperatures and melting points and a fusion enthalpy peak of maximum crystallinity, (88%), which coincided with the point of matrix collapse.     

Aerobic thermophilic bacteria enhance biogas production

The enhancing effect of aerobic thermophilic (AT) bacteria on the production of biogas from anaerobically digested sewage sludge (methanogenic sludge) was investigated. Sewage sludge (5%, w/w) was incubated at 65°C with shaking for a few months to prepare the AT seed sludge. AT sludge was prepared by incubation of the AT seed sludge (5%, v/v) and sewage sludge (5%, w/w) at 65°C with shaking. The addition of this AT sludge (1.2% ± 0.5% of total volatile solids) to methanogenic sludge enhanced the production of biogas. The optimum volume of the addition and the pretreatment temperature of the AT sludge for optimum biogas production were 5% (v/v) and 65°C. Batch-fed anaerobic digestion was covered with the addition of various AT sludges. The AT sludge prepared with the AT seed sludge improved the biogas production by 2.2 times relative to that from the sewage sludge addition. The addition of sludge without AT seed sludge weakly enhanced biogas production. An aerobic thermophilic bacterium (strain AT1) was isolated from the AT seed sludge. Strain AT1 grew well in a synthetic medium. The production of biogas from the anaerobic digestion of sewage sludge was improved by the addition of 5% (v/v) AT1 bacterial culture compared with that from the sewage sludge addition. The addition of AT1 culture reduced the volatile solids by 21%, which was higher than the 12.6% achieved with the sewage sludge addition. The AT1 bacterial culture enhanced the biogas production more than the AT seed sludge. The phylogenetic analysis of the 16S rRNA gene revealed that strain AT1 is closely related to Geobacillus thermodenitrificans (100% sequence similarity). The improvement in the production of biogas with the AT sludge could be caused by thermophilic bacterial activity in the AT sludge.     

Synthesis and environmental degradation of polyesters based on poly (ε-caprolactone)

Poly (-caprolactone) (PCL), poly (-valerolactone) (PVL), poly (-caprolactone-co--valerolactone) [P(CL-co-VL)], and poly (-caprolactone-co-ethylene oxide-co--caprolactone) (PCL-PEO-PCL) were synthesized by ring-opening and diol-initiated polymerization of -caprolactone and -valerolactone. The degradation of the samples by chemical hydrolysis and in a soil burial test was evaluated. It was found that PCL, PVL, and P(CL-co-VL) degrade mainly enzymatically. The rate of degradation depends on their molecular weight, chemical structure, composition, and morphology. PCL-PEO-PCL block copolymers exhibit a repelling effect to the microorganisms in the soil, which depends on the molecular weight and relative amount of PEO block in the copolymer.     

Physical state and biodegradation behavior of starch-polycaprolactone systems

The biodegradation behavior of insoluble crystalline polymers depends on both chemical structure and physical state. The physical state is strongly affected by the molding conditions; moreover the presence of natural hydrophylic substances such as starch can further influence the biodegradation process. This paper examines the biotic and abiotic degradation of thick injection-molded parts, made of pure poly--caprolactone (PCL) at different molecular weights, and of PCL in the presence of starch in the case of a commercial grade of Mater-Bi, produced by Novamont. The abiotic degradation was studied at 25 and 50°C, whereas the biotic degradation was followed in conditions of SCAS (semicontinuous activated sludges) at 25 and 50°C, soil burial, and controlled composting. The physical-chemical modifications provoked at the surface and in the bulk of the samples by the different types of degradation were determined by differential scanning calorimetry, viscometric and gravimetric analysis, scanning electron microscopy, and dynamic mechanical analysis. The mechanical modifications induced by the different environments were followed by tensile tests. It was demonstrated that the presence of starch significantly increases the apparent biodegradation rate of PCL, making even thick parts of ZI01U compatible with the composting process.Paper presented at the Bio/Environmentally Degradable Polymer Society—Third National Meeting, June 6–8, 1994, Boston, Massachusetts.     

Influence of Processing Additives on the Degradation of Melt-Pressed Films of Poly(ε-Caprolactone) and Poly(Lactic Acid)

Melt-pressed films of polycaprolactone (PCL) and poly(lactic acid) (PLA) with processing additives, CaCO₃, SiO₂, and erucamide, were subjected to pure fungal cultures Aspergillus fumigatus and Penicillium simplicissimum and to composting. The PCL films showed a rapid weight loss with a minor reduction in the molecular weight after 45 days in A. fumigatus. The addition of SiO₂ to PCL increased the rate of (bio)erosion in A. fumigatus and in compost. The use of a slip additive, erucamide, was shown to modify the properties of the film surface without decreasing the rate of bio(erosion). Both the rate of weight loss and the rate of molecular weight reduction of PCL increased with decreasing film thickness. The addition of CaCO₃ to PLA significantly reduced the thermal degradation during processing, but it also reduced the rate of the subsequent (bio)degradation in the pure fungal cultures. PLA without additives and PLA containing SiO₂ exhibited the fastest (bio)degradation, followed by PLA with CaCO₃. The degradation of the PLA films was initially governed by chemical hydrolysis, followed by an acceleration of the weight change and of the molecular weight reduction. PLA film subjected to composting exhibits a rapid decrease in molecular weight, which then remains unchanged during the measurement period, probably because of crystallization.     

Accumulation of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid-co-4-hydroxyvaleric acid) by mutants and recombinant strains ofAlcaligenes eutrophus

Alcaligenes eutrophus accumulated a terpolyester of 3-hydroxybutyric acid (3HB), 3-hydroxyvaleric acid (3HV), and 4-hydroxyvaleric acid (4HV) during cultivation with 4HV as carbon and energy source under nitrogen starvation. The polyester accumulated by wild-type strains under these conditions contained 4HV at a molar fraction of approximately 5 mol% only. A catabolic pathway of 4HV was postulated, which included the activation of 4HV to 4HV-CoA and a conversion of 4HV-CoA to 3HV-CoA. Tn5::mob-induced mutants were isolated fromA. eutrophus HF39, which were affected in 4HV and/or valeric acid catabolism. Among 83 mutants were 27 4HV-negative or 4HV-leaky mutants; two mutants were identified which accumulated a terpolyester with a molar fraction of 10.1 to 22.7 mol% 4HV. In addition, a further increase in the molar fraction of 4HV in poly(3HB-co-3HV-co-4HV) and a two- to fourfold increase in the PHA synthase activity were monitored in these mutants or others and also in HF39, if the cells were complemented with the hybrid plasmid pHP1014::PP1, which contained the PHA biosynthesis genes ofA. eutrophus H16. Application of mutagenesis plus recombinant DNA techniques resulted in the accumulation of a terpolyester with up to 30 mol% 4HV and with approximately equimolar fractions of 3HB, 3HV, and 4HV.     

Evaluation of biodegradability of poly(ε-caprolactone)/poly(ethylene terephthalate) blends

The results of an investigation aimed at evaluation of the biodegradability of blends of poly(-caprolactone) (PCL) with poly(ethylene terephthalate) (PET) as the major component are reported. Specimens of the blends, as melt extruded films and/or powders, were submitted to degradation tests under different environmental conditions including full-scale composting, soil burial, bench-scale accelerated aerobic degradation, and exposure to axenic cultures and esterolytic enzymes. Indications have been gained that blending in the melt gives rise to insertion of PCL segments in the PET chain. Copolymers thus attained acted as macromolecular compatibilizers, allowing for a complete miscibility of PCL and PET. The biodegradation detected on the blend samples was, however, well below the values expected from chemical composition and behavior of individual homopolymers under the same environmental conditions. The presence of PET as the major component in PET/PCL blends apparently reduces the propensity of PCL to be degraded, at least in the investigated composition range. The degradation data collected under different environmental conditions indicate that the full-scale composting system is the most efficient among the tested degradation procedures.     

Hydrolytic and enzymatic degradation of poly(γ-glutamic acid) hydrogels and their application in slow-release systems for proteins

The hydrolytic and enzymatic degradation of newly developed hydrogels, produced by cross-linking purified poly(-glutamic acid) (PGA) with dihaloalkane compounds, was studied and is reported in this paper. Analysis of hydrolysis of the hydrogel as a function of pH indicated that the hydrolysis occurred slowly at neutral pH, but fast in both acidic and alkaline solutions, while the polymer could be hydrolyzed rapidly only in acidic solutions. The ester bonds were more sensitive to hydrolysis than peptide bonds. The biodegradability of the hydrogel and polymer was further confirmed when enzymatic degradation was studied by three enzymes (cathepsin B, pronase E, and trypsin), which were able to cleave both ester and peptide bonds gradually. A slow-release system for porcine somatotropin (pST) formed by using the hydrogel as matrix to entrap the hormone was evaluatedin vitro andin vivo. Results demonstrated that the hydrogel was able to release the hormone for a period of 20–30 days and indicated its potential application in slow-release systems for bioactive materials, especially macromolecules, such as peptides and proteins.